The notion that a specific bone marrow (BM) microenvironment promotes the differentiation of lineage- committed blood precursors was suggested 50 years ago by Marcel Bessis where he described in seminal studies the erythroblastic island, a structure made of a central macrophage (M) surrounded by erythroblasts at varying stages of differentiation. Although studies using in vitro reconstitution analyses have clearly suggested a role for marrow-derived M? in erythropoiesis, there is currently no in vivo evidence that M? are required or even play a role in postnatal erythropoiesis. A major reason for the deficit of in vivo knowledge stems from the poorly defined nature of BM M? and, until recently, the lack of specific genetic models. We have defined subsets of BM mononuclear phagocytes and showed that CD169+ M? regulated the hematopoietic stem cell niche. Our preliminary studies also suggest that BM CD169+ M? promote erythropoiesis since their selective in vivo depletion compromised erythropoietic recovery after 5-fluorouracil- or phenylhydrazine-induced anemia. Additionally, in a genetic model of polycythemia vera (PV) from transgenic overexpression of mutated JAK2V617F, we have found that M? depletion normalized the hematocrit, suggesting that erythropoiesis in PV, unexpectedly, depends on signals from the microenvironment. Based on these preliminary results, we propose to evaluate further the molecular basis and function of BM M? in healthy and diseased erythropoiesis. In Specific Aim 1, we will define in vivo molecular mechanisms regulating the BM erythroid niche (erythroblastic island). We will characterize CD169+ erythroblastic islands in collaboration with Drs M. Narla (NYBC) and J. Chasis (Berkeley) and get new insight on central M? by flow cytometry and transcriptional profiling collaboration with Dr. M. Merad (Mt. Sinai). We will generate macrophage-specific genetic models for candidate M? receptors previously suggested to play a role in the erythroblastic island (Vcam1, Itgav, and Maea). In Specific Aim 2, we will examine the contribution of bone marrow M? in chronically diseased erythron. We will analyze models of erythropoietic stress such as sickle cell disease and thalassemia, the latter in collaboration with Dr. S. Rivella (Cornell). We will then evaluate the impact of macrophage in transgenic JAK2V617F models in collaboration with Dr. J. Zhao (Oklahoma) and Tony Green (Cambridge) using M?-specific CD169-DTR depletion and M?-specific genetic models generated in the first aim. In Specific Aim 3, we will examine the potential therapeutic benefit of expanding the central macrophage in the erythropoietic recovery after myeloablation. We will use transgenic models (Csf1 overexpression in collaboration with Dr. R. Stanley, Einstein) and pharmacological approaches (long-acting IL-4 complexes). These studies will define for the first time the in vivo roles of BM macrophages in erythropoiesis and will likely lead to novel ways to regulate erythropoiesis in diseases characterized by erythron expansion. (End of Abstract)